| Objective: To investigate the expression of aquaporin5channel protein and to explore on itsrelationship with apoptosis in well and poorly differentiated non-small cell lungcarcinoma both in-vivo and in-vitro.Methods: The expression of AQP5was detected by immunohistochemistry in lungsquamous and adenocarcinoma tissues, and the apoptosis index determined by the TUNELstaining method. Induction of differentiation in A549cells was done using all-trans retinoicacid (ATRA), and cell proliferation measured by MTT. Reversal of malignancy in A549cellswas measured using the Periodic Acid Schiff Staining Method for mucins and associatedglycoconjugates. The carcinoembryonic (CEA) immunohistochemistry assay was also used tomeasure ATRA induced reversal of malignancy in A549cells. The expression of AQP5wasdetermined using the immunoflorescence assay. ATRA induced apoptosis was measuredusing Annexin V/PI flow cytometry assay, caspase3immunoflorescence and Hoechst33342staining.Results: For squamous cell lung carcinoma，the mean optical density of AQP5of well-differentiated squamous cell carcinoma (0,283±0,02) was significantly higher than thatof poorly differentiated squamous cell lung carcinoma group (0,219±0,05)(p<0.05), whilethe diameter of well-differentiated cancer cells (27,02±0,83μm) was significantly higherthan that of poorly differentiated lung squamous cell carcinoma (17,85±0,82μm)(p<0.05).The apoptosis index (per1000cells) of well differentiated lung squamous cell carcinomagroup (19,93±2,37) was significantly higher than that of poorly differentiated lungsquamous cell carcinoma group (17,27±1,80)(p<0.05). For lung adenocarcinoma， thediameter of well-differentiated adenocarcinoma (13,60±0,82μm) was significantly lowerthan that of poorly differentiated lung adenocarcinoma cells (28,01±1,01μm)(p<0.05),while the mean optical density of AQP5in the well-differentiated group (0,281±0,03) wassignificantly higher than that in poorly differentiated lung adenocarcinoma group (0,238±0,02)(p<0.05). The apoptosis index (per1000cells) of well-differentiated adenocarcinoma group(20,20±2,40) was significantly higher than that in poorly differentiated adenocarcinomagroup (18,53±1,73)(p<0.05). Significant positive correlations were found in both high and low adenocarcinoma tissue groups between mean optical density and the apoptosis index (r=0.524; p=0.045and r=0.661; p=0.007respectively) while no significant correlation wasfound in both high and poorly differentiated squamous cell groups between mean opticaldensity and the apoptosis index (r=-0.069; p=0.807and r=-0.299; p=0.279respectively). Invivo, ATRA decreased cell proliferation in a dose dependent way, and the expression ofaquaporin5was depended on the degree of cellular differentiation. The cytomorphologicalchanges, expression of differentiation markers, caspase3expression and flow cytometryapoptotic results were also dependent on the dose of ATRA treatment. Results for AQP5(mean optical density, MOD) were as follows; control group,1,29±0,14;5μg/ml group,1,31±0,15;10μg/ml group,1,32±0,10and15μg/ml group,1,33±0,12while flow cytometry(apoptotic index, AI) results were; control group,1,01%;5μg/ml group,5,67%;10μg/mlgroup,6,13%and15μg/ml group,7,15%. The increase in the expression of AQP5per cell(mean optical density) with increasing dose of ATRA treatment was significantly correlatedto the increase in the apoptosis of A549cells (r=0.970; p=0.0304).Conclusion: The expression of AQP5both in-vivo and in-vitro is dependent on the type anddegree of tumour differentiation. In-vivo, an increase in aquaporin5expression is associatedwith an increased apoptosis in both poorly and highly differentiated adenocarcinoma whilethere is no association between aquaporin5expression and apoptosis in both poorly andhighly differentiated squamous cell carcinoma. The apoptotic index of adenocarcinoma tissueis higher than that of squamous cell carcinoma. In vitro, differentiation therapy in the form ofATRA decreases both cell proliferation and increases the expression of AQP5in A549cells.The cytomorphological changes, expression of differentiation markers, caspase3expressionand flow cytometry apoptotic results are dependent on the dose of ATRA treatment. Theincrease in AQP5which is associated with an increase in apoptosis was significant in A549cells and this agrees well with findings from the tissue study. This means that in lungadenocarcinoma, a higher expression of aquaporin5promotes the rate of the apoptoticprocess. |
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